Sialidase NEU3 defines invasive potential of human glioblastoma cells by regulating calpain-mediated proteolysis of focal adhesion proteins.

BACKGROUND:Glioblastoma multiforme is one of the most malignant tumors of the human central nervous system characterized by high degree of invasiveness. Focusing on this invasive nature, we investigated whether ganglioside-specific sialidase NEU3 might be involved, because gangliosides are major components of brain tissues, and cell surface sialic acids, as target residues of sialidase catalysis, are thought to be closely related to cell invasion. METHODS:NEU3 mRNA levels of human glioblastoma specimens were evaluated by quantitative RT-PCR. Human glioblastoma cell lines, U251, A172, and T98G were used for cell invasion and migration by transwell and cell scratching assay. The molecules involved in the signaling cascade were investigated by western blot and immunofluorescent microscopy. RESULTS:NEU3 expression was down-regulated in human glioblastoma specimens. In the human glioblastoma cell lines, NEU3 overexpression reduced invasion and migration by promoting the assembly of focal adhesions through reduced calpain-dependent proteolysis, but NEU3 silencing resulted in accelerating cell invasion via disassembly of focal adhesions. In NEU3-silenced cells, elevation of calpain activity and GM3 accumulation were observed, as results of reduced sialidase hydrolysis, localization of calpain and GM3 at the cell lamellipodium being demonstrated by immunofluorescence microscopy. CONCLUSION:Sialidase NEU3 was found to exert a great influence on cell invasion in regulation of calpain activity and focal adhesion disassembly and consequent invasive potential of glioblastoma cells. GENERAL SIGNIFICANCE:This first demonstration of sialidase involvement in invasive potential of gliolastoma cells may point to NEU3 as an attractive treatment target of human gliomas.